POS0499 A NEW SERUM ASSAY MEASURING SYNOVIAL TURNOVER IN RHEUMATOID ARTHRITIS
Authors: P. Garnero, E. Gineyts, J. C. Rousseau, H. Marotte, R. Chapurlat
Journal: Annals of the Rheumatic Diseases (2022)

Background Rheumatoid arthritis (RA) is characterized by synovial tissue (ST) inflammation leading to pain, joint destruction, impaired mobility and ultimately increased morbidity and mortality. These various components of disease etiology can be monitored by pain scores and imaging technologies. Noninvasive assessment of joint tissues metabolism can be performed by measuring serum metabolites of tissue matrix turnover. Specific biochemical markers (BM) of bone and cartilage have been developed, but there is still a lack of a sensitive index of ST metabolism abnormalities, although there are early and key drivers of joint destruction. Objectives To develop a sensitive ELISA-based serum BM of ST turnover (SynHelix) and evaluate its performance in patients with RA. Methods We identified a 11 amino-acid sequence (HELIX-III) within the triple helical portion of type III collagen, a main component of ST matrix which is released during ST turnover and can be measured in the serum. A highly specific rabbit polyclonal antibody raised against the synthetic HELIX-III peptide was produced to develop a competitive ELISA. Specificity of the antibody was evaluated by competitive inhibitions with homologous sequences of type I, IV and XI collagens which are also present in ST, but in minor quantities for the two later. Immunohistochemistry analysis of human ST obtained during hip surgery was performed to investigate in situ localisation of the HELIX-III peptide. ELISA was then used to quantify serum HELIX-III fragments in two samples of patients with low (n= 40, median DAS 28: 2.1) and moderate (n=11, median DAS28: 3.5) RA activity participating in clinical trials. Levels in RA subjects were compared to sex and age-matched healthy controls randomly selected from epidemiological cohorts (n=70). Results The ELISA for SynHelix demonstrated adequate analytical performances with intra and interassay variations below 10 and 15%, respectively; analytical and functional limits of quantification of 0.21 and 2.5 ng/ml, respectively and dilution recovery of human serum ranging from 80 to 108%. Competitive inhibition experiments demonstrated that the antibody does not recognize HELIX-III peptide shortened or elongated by one amino-acid, indicating that immunogenicity is dependent on the presence of a neopitope resulting from the cleavage of the collagen molecule. The antibody does not recognize the homologous sequence of type I collagen, but shows significant immunoreactivity with the homologous sequences of type IV (alpha 5) and type XI (alpha 2) collagens with however a lower affinity than for type III collagen (a 2.2 and 4-fold higher concentration, respectively, is needed to displace 50% of the immune signal). Immunohistochemistry of ST tissue from RA subjects showed increased staining in the interstitial tissue and around vessels which are rich in type IV collagen. Median serum levels of SynHelix were significantly higher in patients with low (p=0027, +17%) and moderate RA activity (+220%, p=00004 vs healthy controls; +164% p=0.16 vs low RA) compared with those in age-matched controls, although CRP did not discriminate RA patients with low and moderate activity (2.2 mg/L for both groups, p=0.90). In RA subjects, serum SynHelix correlates modestly with CRP (r=0.59, p &lt;0.0001), but not with DAS28. Conclusion The new SynHelix ELISA measures precisely circulating degradation fragments of Helix-III peptide-containing collagens. Serum SynHelix levels are already increased in patients with low activity RA, values correlating modestly the degree of systemic inflammation. Larger longitudinal studies are needed to further evaluate the value of SynHelix to predict disease outcome in RA. Disclosure of Interests Patrick Garnero: None declared, Evelyne Gineyts: None declared, Jean-Charles Rousseau: None declared, Hubert MAROTTE: None declared, Roland Chapurlat Consultant of: BMS, abbvie, Lilly, Galapagos, fresenius-Kiabi, novartis.